Knowledge of the extent of direct bonding interactions between the activating metal ion and potential ligand donor groups of the substrate at the active site of an enzyme is essential to an understanding of the role of the metal ion in the catalytic events. Enzymes which use metal-nucleotide complexes as substrates or effectors are of central importance in a wide variety of cellular processes. Enzymes which use metal complexes with ATP or GTP from several functional categories such as kinases, ligases, nucleotidyl transferases, and ATPases will constitute the major focus of the investigation. The proposed research will use spectroscopic methods such as nuclear magnetic resonance, electron paramagnetic resonance, and infrared spectroscopy which are capable of detecting direct metal-ligand bonding at the active site or regulatory sites of an enzyme. Thus, superhyperfine coupling between magnetically active nuclei such as 1/0 and the unpaired electron spins of the manganous ion can be observed in electron paramagnetic resonance spectra for the enzymic complexes, and such observations provide unambiguous evidence for direct bonding. Stereospecific incorporation of the magnetically active nuclei into the substrate molecule will permit identification of the stereochemical configuration of the enzyme-bound metal-substrate complex. Metal ion specific perturbations in the absorption frequencies and in the multiplicities of infrared absorptions of the bound substrate will also be used to identify direct metal substrate interactions. The structural data will be correlated with possible functional roles of the metal ions in the catalytic or regulatory activities.